1. Field of the Invention:
The present invention relates to a method for increasing cellular levels of glutathione.
2. Description of the Prior Art:
It is well-known that the tripeptide thiol glutathione (L-.gamma.-glutamyl-L-cysteinyl-glycine; GSH) found in virtually all cells functions in metabolism, transport and cellular protection. Glutathione functions in the reduction of the disulfide linkages of proteins and other molecules, in the synthesis of the deoxyribonucleotide precursors of DNA, and in the protection of cells against the effects of free radicals and of reactive oxygen intermediates such as peroxides that are formed in metabolism.
Modifications of glutathione metabolism may be achieved by administration of selective enzyme inhibitors to decrease intracellular glutathione levels, or by providing compounds that increase glutathione synthesis. Such effects are useful in chemotherapy and radiation therapy and in protecting cells against the toxic effects of drugs, other foreign compounds and oxygen. Indeed, the diverse functions of GSH are relevant to many fields of biology, including not only enzymology and transport but also pharmacology, radiation biology, cancer therapy, toxicology, endocrinology, microbiology and agriculture. The enzymatic and transport phenomena of glutathione metabolism are outlined in Meister. "Selective Modification of Glutathione Metabolism", Science, Volume 220, Number 4596, 472-477 (April 1983), which is hereby incorporated by reference.
Modification of GSH metabolism to deplete or increase cellular GSH may serve various purposes. For instance, it has long been known that thiols protect cells against the effects of irradiation. Since decreasing cellular GSH makes cells more susceptible to irradiation, glutathione depletion is useful in chemotherapeutic situations in which the cells to be killed and the cells to be spared have substantially different quantitative requirements for GSH. Depletion of GSH by inhibition of its synthesis also serves as a valuable adjuvant in chemotherapy with drugs that are detoxified by reactions involving GSH.
Conversely, development of resistance to a drug or to radiation may be associated with an increase in cellular GSH. GSH serves effectively in the detoxification of many drugs, and it is known that a significant pathway of acetaminophen detoxification involves conjugation with GSH.
Treatment with a thiazolidine such as L-2-oxothiazolidine-4-carboxylic acid, may be of value to patients with liver disease and to premature infants who may be deficient in the utilization of methionine sulfur for cysteine formation, and thus in GSH synthesis. The effectiveness of such a thiazolidine as an intracellular cysteine precursor depends on the presence of 5-oxoprolinase, an enzyme activity found in almost all animal cells. This enzyme also occurs in plants, suggesting that such a thiazolidine, and hence glutathione, may be useful as a safener in agriculture to protect crop plants against the toxic effects of herbicides.
Various methods are known to increase cellular levels of glutathione. Glutathione is composed of three amino acids: glutamic acid, cysteine and glycine. Administration to animals of the amino acid precursors of glutathione may produce an increase in cellular glutathione, but there is a limit to the effectiveness of this procedure. Concentrations of GSH are dependent on the supply of cysteine, which is derived from dietary protein and by trans-sulfuration from methionine in the liver. Administration of cysteine is not an ideal way to increase GSH concentration because cysteine is rapidly metabolized and furthermore, it is very toxic. Administration to animals of compounds that are transported into cells and converted intracellularly into cysteine is sometimes useful in increasing cellular glutathione levels. For example, the thiazolidine L-2-oxothiazolidine-4-carboxylate is transported into the cell, where it is converted by 5-oxoprolinase to L-cysteine, which is rapidly used for GSH synthesis.
Another way in which tissue GSH concentration may be increased is by administration of .gamma.-glutamylcysteine or of .gamma.-glutamylcystine. The administered .gamma.-glutamyl amino acid is transported intact and serves as a substrate of GSH synthetase. It is also known that administration of N-Acetyl-L-cysteine increases tissue concentrations of GSH.
That the administration of glutathione itself might lead to increased glutathione levels has also been considered. However, there is no published evidence that shows that intact glutathione enters cells. In fact, there are several reports on particular biological systems indicating that glutathione itself is not transported into cells. The increase in cellular glutathione sometimes found after administration of glutathione is due to (a) extracellular breakdown of glutathione, (b) transport into cells of free amino acids or dipeptides derived from glutathione extracellularly, and (c) intracellular resynthesis of glutathione.
These previous methods of increasing intracellular glutathione concentration are disadvantageous in the areas of efficiency, toxicity, limits on effective concentration obtainable, etc. as discussed heretofore. In addition, the known methods which depend on synthesis of GSH by increasing the supply of substrates to the two synthetases involved, depend on the presence of the synthetases, the first of which is subject to feedback inhibition by GSH.
Accordingly, an object of the invention is to provide a method for increasing the intracellular levels of glutathione by delivering intact glutathione to the cell rather than its amino acid substrates.
A further object of the invention is to provide pure derivatives of glutathione, the use thereof in glutathione delivery and a method for obtaining such pure derivatives.
Another object of the invention is to provide a method of increasing intracellular levels of glutathione which is highly efficient and which does not depend on the presence of synthetases.
A further object of the present invention is to provide a method for increasing the intracellular levels of glutathione without the toxic effects of other known methods.
Still another object of the invention is to provide a method for efficiency and rapidly increasing cellular glutathione levels for any purpose for which elevated glutathione levels are desired in the prior art, such as for drug detoxification, cellular protection against oxygen and its metabolites such as peroxides, free radicals, or foreign compounds, etc.